CN100465279C - Prepn and application of epiderm or hair follicle stem cell from souce of human early embryo - Google Patents
Prepn and application of epiderm or hair follicle stem cell from souce of human early embryo Download PDFInfo
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- CN100465279C CN100465279C CNB2006100253176A CN200610025317A CN100465279C CN 100465279 C CN100465279 C CN 100465279C CN B2006100253176 A CNB2006100253176 A CN B2006100253176A CN 200610025317 A CN200610025317 A CN 200610025317A CN 100465279 C CN100465279 C CN 100465279C
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Abstract
This invention provides a method for accelerating the proliferation of human mesenchymal stem cells, which comprises the steps of: transfecting recombinant expression carrier containing EGF to human mesenchymal stem cells, and stimulating with a certain concentration of calcium ions during the transfection process to divide into typical stem cells. Under nude mouse subcutaneous microenvironment induction, the stem cells are further divided into skin tissue stem cells, thus directly taking part in the proliferation of nude mouse hair. This invention can obtain rapidly proliferated human mesenchymal stem cells, thus having a potential application in clinical skin wound treatment.
Description
Technical field
The present invention relates to stem cell and gene engineering technology field, be specifically related to method and the application thereof of a kind of people's embryo source property epidermis (hair follicle) stem cell that can fast breeding.
Background technology
Skin is the largest organ of human body, because of being in body surface, suddenly is vulnerable to damage.Very common clinically as burn, scald, contusion, wound etc.The covering of skin wound and the regeneration of epidermic cell are the keys of treatment skin injury.The healing that at all is the surface of a wound of trauma care.At present existing the big area degree of depth surface of a wound still adopted auto-skin grafting, restorative procedure such as more plant from/allosome mixed transplantation or from/heterogenous skin, these methods exist the course of treatment long, complication is many, medical expense is high, healing quality is poor, cause new shortcomings such as the surface of a wound.In the skin wound repair process, the regeneration of cutaneous appendages is a difficult problem.Along with going deep into of stem-cell research, people attempt carrying out Transplanted cells and treat skin wound.But the epidermal stem cells propagation in adult source slowly, the culture condition harshness.
1962, Stanley Cohen was a kind of small molecules single chain polypeptide at female mice lower jaw gland table of discovery skin growth factor (EGF).1975, Harry Gregory found people's EGF.Except that the lower jaw gland, human body multiple organized also has EGF to distribute, as duodenal Brunners gland, contain neutral mucinous gastric mucosal cell system, external secretion body of gland and kinds of tumor cells etc.EGF can cause endogenic focal adhesion kinase (FAK) phosphorylation of 3T3 cell by extracellular signal-regulated kinase (ERK) approach.Thereby activating cells sticks.The EGF acceptor not only has function of receptors, and has enzymic activity, thus the characteristics that existing binding partner occurred conformation changes, again can be as kinases, catalytic substrate albumen generation tyrosine phosphorylation.Existing known EGF plays a role by combining with EGF acceptor (EGFR) on the cytolemma.EGFR is that a kind of relative molecular mass is the glycoprotein of 170kD, is made of 1186 amino-acid residues, and cerbB1 is coded for proto-oncogene.This gene is positioned on the Sub_clause 11 karyomit(e) of mouse and people's the 7th karyomit(e).Its structure is divided into 3 zones: stretch to film outer with EGF identification also the bonded amino acid region, be positioned at intracytoplasmic carboxyl petiolarea (the Wiley LM that the cytolemma intermediary is striden the film district and had tyrosine kinase activity, Adamson ED, Tsark EC.Epidermal growth factor receptor function in earlymammalian development[J] .Biol Essays.1995; 17 (10): 839-846.).The 26S Proteasome Structure and Function of EGFR is similar substantially in the different genera animal, and molecular evolution is more conservative.EGF can cause that the protein tyrosine kinase activity of EGFR in the cytoplasm increases during with its receptors bind, the phosphorylation of tyrosine residues is increased, thereby cause Cytoplasmic Ca
2+Increase, genetic transcription increases, and dna replication dna increases and cell fission; Ca in cell
2+When increasing, can also activated protein kinase C and the ring adenosinase, change the skeleton structure of cell, make cytodifferentiation.Ga
2+The cell adhesion that relies on can activate CDC42, may be by c-Src and FRG approach (Tatsuro Fukuharal, Kazuya Shimizul, Tomomi Kawakatsul, et al.Activation of Cdc42 by transinteractions of the cell adhesion molecules nectins through c-Src andCdc42-GEF FRG The Journal of Cell Biology.2004; 166:393-405).The intrusion of the shortage of E-calcium adhesion molecule or the change of structure and human specific tumour and lapsed to confidential relation.Discover with the human keratinocyte who cultivates, the formation of intercellular adhesion is that extracellular Ca2 ionic concentration participates in regulation and control, when the calcium ion concn of nutrient solution when low-level (30 μ M), cell is monolayer growth and does not have adhesion function, when calcium ion concn increases (1.0mM), cause the formation that adheres to connection and desmosome fast, so be the attachment proteins (Wang Yuntao that a kind of calcium ion relies on, Zheng Qixin, Guo Xiaodong, etc. wild-type Smad3 gene promotes the experimental study of rat bone marrow mesenchymal stem cells Osteoblast Differentiation. Chinese Medical Journal .2004; 84:1523-1527).
Under specific inside and outside microenvironment condition, stem cell can be induced to differentiate into various histocytes, for multiple treatment of diseases has been expanded new treatment approach.Stem cell is divided into the different tissues cell at different sites, and tissue microenvironment such as calcium ion concn have played vital role on regulation and control differentiation of stem cells direction.
The inventor successfully from 4 to 6 all people from left and right sides embryo separation and Culture people's embryo source property mescenchymal stem cell (hMSC).(coroner is quick beautiful, Liu Houqi etc. and the location of human body early embryo mescenchymal stem cell and external initial gross separation are cultivated. The 2nd Army Medical College journal 2004; 25 (8): 818-821)
Summary of the invention
The objective of the invention is to overcome the adult epidermal stem cells and breed problem slowly, utilize above-mentioned people's embryo source property mescenchymal stem cell (hMSC), method and the application thereof of a kind of people's embryo source property epidermis (hair follicle) stem cell that can fast breeding are provided.
The present invention induces the mescenchymal stem cell (hMSC) in human body early embryo source and is people's embryo source property epidermis (hair follicle) stem cell under particular organization's microenvironment.
The present invention can activate FAK according to the EGF plasmid transfection, changes the skeleton structure of cell, makes cytodifferentiation; The external source calcium ion stimulates can activate this fact of adhesion protein that calcium ion relies on, obtain from people's adult face tissue according to literature method and to contain the EGF gene cDNA fragment and the carrier for expression of eukaryon pCDNA3.0 that recombinates, (with reference to Chen Ting speed etc., synthesizing of human epidermal growth factor (hEGF) gene, the structure of evaluation and plant expression plasmid, Guangxi University's journal (natural science edition), 27 (2) 122-127), transfection people embryo source property mescenchymal stem cell (hMSC), so that with the exogenous EGF protein promoter signal transduction of expressing, promote it to epidermal differentiation, stimulate in conjunction with certain density calcium ion simultaneously.
The invention provides the method for a kind of people's embryo source property epidermis (hair follicle) stem cell that can fast breeding, it comprises and will contain the recombinant expression vector transfection people embryo source property mescenchymal stem cell of EGF, and the calcium ion with 0.4-1.0mmol concentration stimulates in transfection process.
Discovering that by experiment in vitro cellular form changes after the transfection, is short circular cell (Fig. 1) by fiber deformation.After adding the stimulation of finite concentration calcium ion, cell becomes typical epidermic cell shape growth (Fig. 2), and express keratin, Keratin sulfate 19 antigens such as grade (Fig. 3).
Consider the inducing action of the intravital microenvironment of animal to differentiation of stem cells, it is subcutaneous that the present invention plants nude mice with people's embryo source property mescenchymal stem cell.Under the inducing of subcutaneous microenvironment, people's embryo derived stem cells breaks up to epidermic cell.Experiment shows in the body, and people's embryo source property mescenchymal stem cell is at the subcutaneous epidermic cell that is divided into of nude mice, and has participated in the regeneration (Fig. 4) of nude mice hair directly.The result of human keratinous 19 dyeing and alkaline phosphatase staining also shows the skin fine structure of transplanting place of people's embryo source property mesenchymal stem cell transplantation nude mice, has the characteristic (Fig. 5) of epidermis (hair follicle) stem cell.
The method of a kind of people's embryo source property epidermis (hair follicle) stem cell that can fast breeding provided by the invention can obtain propagation people's embryo source property epidermic cell relatively rapidly, can be applicable to clinical skin wound treatment.
Description of drawings
Fig. 1 is the people's embryo source property mescenchymal stem cell behind the transfection EGF
Fig. 2 is people's embryo source property mescenchymal stem cell of the post-stimulatory transfection EGF of calcium ion
Fig. 3 detects the test-results of Keratin sulfate and Keratin sulfate 19 for immunohistochemical methods
A:EGF wherein; B: Keratin sulfate 19; C: Keratin sulfate; D: negative control
Fig. 4 is the burnt detection of immunofluorescence copolymerization
Property mesenchymal cell in source is divided into the test-results of hair in nude mouse
A wherein: the relevant dissolubility antigen HLAI of tissue; B: Keratin sulfate;
C: negative control; D:A and B stack
Fig. 5 behaves, and K Keratin sulfate 19 dyes and the nude mice test-results of alkaline phosphatase staining
C wherein, G: human keratinous 19 coloration results;
D, H: alkaline phosphatase staining result.
Embodiment
Now reach accompanying drawing in conjunction with the embodiments, the invention will be further described.
Embodiment 1:
(1) the total RNA of preparation human epidermal cell:
Become the human body skin through former be commissioned to train foster obtain epidermic cell (Yang Ling, Liu Houqi etc.Become the location and the separation and Culture of human body skin epidermal stem cells.The The 2nd Army Medical College journal, 2004,25 (8): 815-817.).With Shanghai China Shun biotechnology company limited total RNA extraction agent box, guanidine isothiocyanate method routinely extracts and obtains total RNA.
(2) synthetic primer and structure pCDNA3.0-EGF carrier for expression of eukaryon:
The hEGF gene is moved to eukaryotic vector pCDNA3.0.According to document obtain contain the EGF gene cDNA fragment and the carrier for expression of eukaryon pCDNA3.0 that recombinates (with reference to Chen Ting speed etc., synthetic, the evaluation of human epidermal growth factor (hEGF) gene and the structure of plant expression plasmid, Guangxi University's journal (natural science edition), 27 (2) 122-127).
(3) set up pCDNA3.0 and pCDNA3.0-EGF recombinant human embryo source property mescenchymal stem cell cell strain:
Operate by the lipofectamine box specification sheets that U.S. GibcoL produces, with people's embryo source property mescenchymal stem cell of empty carrier pCDNA3.0 and recombinant vectors pCDNA3.0-EGF difference transfection logarithmic phase.Concrete grammar is as follows: long people's embryo source property mescenchymal stem cell to 50%～60% in 6 orifice plates is washed once with serum-free DMEM substratum, add the 2ml serum free medium and cultivated 6 hours.
The preparation of transfection liquid: two liquid below the preparation (for the used amount of each porocyte of transfection) A liquid in polystyrene tube: respectively empty carrier and recombinant vectors are diluted to 1～1o μ g with the DMEM substratum that does not contain serum, every duplicate samples is 100 μ l; B liquid: with the DMEM substratum that does not contain serum plasmalogen is carried out the two-fold dilution, total amount is two 100 μ l; With A, B two liquid are mixing gently, mid-10～15 minutes of room temperature.
Transfection is prepared: do not contain twice in serum DMEM substratum rinsing cell with 2ml; Add 1ml again and do not contain blood serum medium.
Transfection: slowly add in the substratum with the A/B mixture, shake up, place 37 ℃, the incubator of 5%CO2 to cultivate 5 hours, absorb serum-free transfection liquid, change to the DMEM nutrient solution that normally contains 10% calf serum and continue to cultivate.With G418 (U.S. GibcoL) screening (G418 600 μ g/ml), select G418 positive monoclonal cell amplification and cultivate.Get people's embryo source property mescenchymal stem cell cell strain of empty carrier pCDNA3.0 and recombinant plasmid pCDNA3.0-EGF respectively.
(4) calcium ion and EGF coordinative role
Get 55.4g CaCl
2Be dissolved in the 800ml redistilled water, add redistilled water and be settled to 1000ml and prepare 1mol CaCl
2(calcium chloride).Room temperature preservation behind the autoclaving.With containing in culture medium culturing 6 orifice plates 0.4mmol calcium ion, 10%DMEM long people's embryo source property mescenchymal stem cell to 60%～70% transfection EGF.
(5) cell experiment (experiment in vitro)
Utilize biological experimental methods such as immunohistochemical methods, cell counting, flow cytometer, from aspect analysis of cells metamorphosis such as morphology, protein expressions, and the expression of epidermal surface index angle albumen and Keratin sulfate 19 and EGF.
Concrete grammar is as follows:
1) comparison of cell growth state
Observing transfection with inverted phase contrast microscope has pCDNA30-EGF, people's embryo source property mescenchymal stem cell of pCDNA3.0 empty carrier and the people's embryo source property mescenchymal stem cell that does not carry out transfection.As seen people's embryo source property mescenchymal stem cell of transfection pCDNA3.0-EGF is compared cell with other two groups and is obviously become round.Inoculating transfection respectively has pCDNA3.0-EGF, and pCDNA3.0 empty carrier and people's embryo source property mescenchymal stem cell of not carrying out transfection carry out cell counting in 6 orifice plates.Respectively these three kinds of cells are inoculated in 6 orifice plates by 1 * 105/ml, counting was 1 time in per 12 hours, amounted to 96 hours.Count 3 times, average, draw growth curve.The result shows: the people's embryo source property mescenchymal stem cell that contains the pCDNA3.0-EGF recon is compared cell proliferation with the people's embryo source property mescenchymal stem cell that does not carry out transfection is not had considerable change.
After containing 0.4mmol calcium ion culture medium culturing, cell becomes typical epidermic cell feature.
2) immunohistochemical methods detects the expression of Keratin sulfate and Keratin sulfate 19 and EGF
The preparation transfection has pCDNA3.0-EGF, and people's embryo source property mescenchymal stem cell of pCDNA3.0 empty carrier reaches the cell climbing sheet of the people's embryo source property mescenchymal stem cell that does not carry out transfection, and collected cell is containing the cultivation of 0.4mmol calcium ion.Detect the expression level of Keratin sulfate and Keratin sulfate 19 and EGF with immunohistochemical methods.One anti-is the anti-mouse Keratin sulfate of rabbit, Keratin sulfate 19 polyclonal antibodies (Beijing Zhong Shan Bioisystech Co., Ltd) and the anti-people EGF of rabbit polyclonal antibody (SANTA CRUZBIOTECHNOLOGY, INC.), two anti-are the anti-rabbit igg of FITC labelled goat (Beijing Zhong Shan Bioisystech Co., Ltd).The result: after calcium ion stimulates, pCDNA3.0-EGF recombinant human embryo source property mescenchymal stem cell cell strain express keratin and Keratin sulfate 19 and EGF.This explanation pCDNA3.0-EGF recombinant human embryo source property mescenchymal stem cell begins to express specific antigens Keratin sulfate and the Keratin sulfate 19 and the EGF of epidermic cell after calcium ion stimulates.
(6) in vivo test
Test used nude mice BALB/c Nu strain, the SPF level.About 15～the 25g of body weight, 3～4 ages in week are available from the Shanghai Experimental Animal Center.Totally 24 nude mices are divided four groups: people's embryo source property mescenchymal stem cell injection group, people's adult skin flbroblast injection group, serum-free DMEM injection group, wild group.With people's embryo source property mescenchymal stem cell and people's adult skin flbroblast is that mother liquor is prepared into total cellular score and is not less than 1 * 10 with serum-free DMEM substratum respectively
7The cell suspension of individual/0.2ml, it is subcutaneous to extract cell suspension 0.2ml injection nude mice back with the 1ml syringe.Feed under the SPF level condition, observe every day, draws materials after all around, found that: injection cell place hairiness becomes.Each treated animal is after experiment is handled, at aspect indifferences such as profile, activity.Respectively organize between the nude mice after the execution and compare, H-E dyeing. internal organs indifferences such as liver,spleen,kidney.
The skin histology of each group nude mice injection site is done Keratin sulfate 19 dyeing, and microscopically is observed and found: the nude mice skin of injection people embryo source property mescenchymal stem cell has a large amount of hair follicles to occur, and other each group does not then have this phenomenon.Alkaline phosphatase staining shows clearly also that the nude mice of injection people embryo source property mescenchymal stem cell is subcutaneous has a large amount of hair follicles to generate.
Concrete grammar is as follows:
1) fluorescence co-focusing
Each group experiment nude mice injection place skin biopsy of preparation.With burnt relevant melting property antigen HLA-I of tissue and the keratic expression level of detecting of immunofluorescence copolymerization.One anti-is mouse anti human monoclonal antibody HLA-I (Sigma CO) and the anti-mouse polyclonal antibody of rabbit Keratin sulfate (Beijing Zhong Shan Bioisystech Co., Ltd).Two anti-are anti-mouse IgG of FITC labelled goat (magnificent biotechnology company limited product) and the anti-rabbit igg of TRITC labelled goat (Beijing Zhong Shan Bioisystech Co., Ltd).The result: the skin of people's embryo source property mescenchymal stem cell injection place detects relevant melting property antigen HLA-I of a large amount of tissues and Keratin sulfate.Both expression overlap.This explanation people embryo source property mescenchymal stem cell breaks up not hair follicle in nude mouse, caused the regeneration of nude mice hair.
2) hematoxylin-eosin (HE) dyeing
Method is with cut into slices hematoxylin-eosin (HE) dyeing of routine paraffin wax.
3) alkaline phosphatase staining
Method is as follows: the frozen section of each experimental group nude mice skin of prepared fresh is taken out, and 2% Paraformaldehyde 96 is fixed, and TSM1 washes, and TSM2 washes, the NBT/BCIP colour developing.
Claims (2)
1, the method for a kind of fast breeding people embryo source property epidermal stem cells or hair follicle stem cells, it is characterized in that to contain the mescenchymal stem cell in the recombinant expression vector transfection human body early embryo source of Urogastron, it is induced be people's embryo source property epidermal stem cells or hair follicle stem cells, and the calcium ion with 0.4-1.0mmol concentration stimulates in transfection process.
2, the method for a kind of fast breeding people embryo source property epidermal stem cells according to claim 1 or hair follicle stem cells, the recombinant expression vector that it is characterized in that Urogastron wherein is carrier for expression of eukaryon pCDNA3.0-EGF.
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| CN103194425B (en) * | 2013-04-02 | 2014-12-31 | 中国人民解放军第二军医大学 | Method for promoting epidermal cell proliferation |
| CN107201341B (en) * | 2017-07-12 | 2020-05-26 | 广州润虹医药科技股份有限公司 | Preparation method of hair follicle stem cells |
| CN112704728A (en) * | 2020-11-09 | 2021-04-27 | 东莞清实生物科技有限公司 | Preparation method and application of EGF (epidermal growth factor) -high-expression stem cell temperature-sensitive gel |
| CN114958733A (en) * | 2021-07-29 | 2022-08-30 | 北京博百悦生物技术有限公司 | Preparation method of autologous hair follicle mesenchymal stem cells |
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| CN1590538A (en) * | 2003-09-02 | 2005-03-09 | 中国人民解放军第四军医大学口腔医学院 | Separation and culturing method of human epidermis stem cell |
| WO2005040391A1 (en) * | 2003-10-27 | 2005-05-06 | Murdoch Childrens Research Institute | Compositions and methods for differentiating stem cells |
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| CN1590538A (en) * | 2003-09-02 | 2005-03-09 | 中国人民解放军第四军医大学口腔医学院 | Separation and culturing method of human epidermis stem cell |
| WO2005040391A1 (en) * | 2003-10-27 | 2005-05-06 | Murdoch Childrens Research Institute | Compositions and methods for differentiating stem cells |
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